Cross-linked glycopeptide-cephalosporin antibiotics

ABSTRACT

This invention provides cross-linked glycopeptide—cephalosporin compounds and pharmaceutically acceptable salts thereof which are useful as antibiotics. This invention also provides pharmaceutical compositions containing such compounds; methods for treating bacterial infections in a mammal using such compounds; and processes and intermediates useful for preparing such compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/486,484, filed on Jul. 11, 2003; the entire disclosure of which isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention is directed to novel cross-linkedvancomycin—cephalosporin compounds which are useful as antibiotics. Thisinvention is also directed to pharmaceutical compositions comprisingsuch compounds; methods of using such compounds as antibacterial agents;and processes and intermediates for preparing such compounds.

2. State of the Art

Various classes of antibiotic compounds are known in the art including,for example, β-lactam antibiotics, such as cephalosporins, andglycopeptide antibiotics, such as vancomycin. Cross-linked antibioticcompounds are also known in the art. See, for example, U.S. Pat. No.5,693,791, issued to W. L. Truett and entitled “Antibiotics and Processfor Preparation”; and WO 99/64049 A1, published on Dec. 16, 1999, andentitled “Novel Antibacterial Agents.” Additionally, WO 03/031449 A2,published on Apr. 17, 2003, and entitled “Cross-LinkedGlycopeptide—Cephalosporin Antibiotics” discloses compounds having aglycopeptide group covalently linked to the oxime moiety of acephalosporin group.

Due to the potential for bacteria to develope resistance to antibiotics,however, a need exists for new antibiotics having unique chemicalstructures. Additionally, a need exists for novel antibiotics havingimproved antibacterial properties including, by way of example,increased potency against Gram-positive bacteria. In particular, a needexists for new antibiotics that are highly effective againstantibiotic-resistant strains of bacteria, such as methicillin-resistantStaphylococci aureus (MRSA).

SUMMARY OF THE INVENTION

The present invention provides novel cross-linkedglycopeptide—cephalosporin compounds that are useful as antibiotics. Thecompounds of this invention have a unique chemical structure in which aglycopeptide group is covalently linked to a pyridinium moiety of acephalosporin group. Among other properties, compounds of this inventionhave been found to possess surprising and unexpected potency againstGram-positive bacteria including methicillin-resistant Staphylococciaureus (MRSA).

Accordingly, in one aspect, the invention provides a compound of formulaI:

or a pharmaceutically-acceptable salt thereof; wherein

each of X¹ and X² is independently hydrogen or chloro;

W is N or CCl;

R¹ and R² are independently selected hydrogen and C₁₋₆ alkyl;

each R³ is independently selected from C₁₋₆ alkyl, OR, halo, —SR,—S(O)R, —S(O)₂R, and —S(O)₂OR, where each R is independently C₁₋₆ alkyloptionally substituted with COOH or 1 to 3 fluoro substituents;

one of R⁴ and R⁵ is hydroxy and the other is hydrogen;

R⁶ and R⁷ are independently hydrogen or methyl;

R⁸ is hydrogen or a group of the formula:

R⁹ is selected from hydrogen, C₁₋₆ alkyl and C₃₋₆ cycloalkyl, wherealkyl and cycloalkyl are optionally substituted with —COOH or 1 to 3fluoro substituents;

R^(a) is —Y—R″—, where R″ is selected from C₁₋₁₂ alkylene, C₂₋₁₂alkenylene, C₂₋₁₂ alkynylene, C₃₋₆ cycloalkylene, C₆₋₁₀ arylene, C₂₋₉heteroarylene, C₃₋₆ heterocycle and combinations thereof, and isoptionally substituted with 1 or 2 groups selected from Z, where each Zis independently selected from —OR′, —SR′, —F, —Cl, —N(R′)₂, —OC(O)R′,—C(O)OR′, —NHC(O)R′, —C(O)N(R′)₂, —CF₃, —OCF₃, and side chains ofnaturally-occurring amino acids, where each R′ is independently hydrogenor C₁₋₄ alkyl; and R″ contains at most 20 non-hydrogen atoms; and Y,which links R″ to the pyridinium ring at a meta or para position, isselected from the group consisting of a direct bond, NR′, O (ether), S(sulfide), C(O) (carbonyl), NR′C(O), and C(O)NR′, precluding directbonds between heteroatoms in Y and R″;

each R^(b) and R^(d) is independently selected from hydrogen, C₁₋₆alkyl, C₂₋₆ alkenyl and C₂₋₆ alkynyl;

each R^(c) is independently a direct bond or —Y′—R″—Y′—, where each Y′is independently selected from a direct bond, O (ether) and NR′,precluding direct bonds between heteroatoms in Y′ and R″;

each R^(e) is independently selected from the group defined by R″ above;

n is an integer from 0 to 3;

x is an integer from 0 to 2; and

y is an integer from 0 to 2.

In another of its composition aspects, this invention provides acompound of formula

or a pharmaceutically acceptable salt thereof; wherein

W is N or CCl;

R⁹ is selected from hydrogen, C₁₋₆ alkyl and C₃₋₆ cycloalkyl, wherealkyl and cycloalkyl are optionally substituted with —COOH or 1 to 3fluoro substituents;

the pyridinium ring has meta or para substitution;

R¹⁰ is hydrogen, C₁₋₄ alkyl or C₂₋₄ alkenyl;

R¹¹ is C₁₋₁₂ alkylene or C₂₋₁₂ alkenylene; and

R¹² is hydrogen, C₁₋₄ alkyl or C₂₋₄ alkenyl.

In another of its composition aspects, this invention provides apharmaceutical composition comprising a pharmaceutically-acceptablecarrier and a therapeutically effective amount of a compound of formulaI, or a pharmaceutically-acceptable salt thereof; including any of theparticular embodiments discussed herein.

The compounds of this invention are useful as anti-bacterial agents.Accordingly, in one of its method aspects, this invention provides amethod of treating a bacterial infection in a mammal, the methodcomprising administering to a mammal a pharmaceutical compositioncomprising a pharmaceutically-acceptable carrier and a therapeuticallyeffective amount of a compound of formula I, or apharmaceutically-acceptable salt thereof; including any of theparticular embodiments discussed herein.

While not intending to be limited by theory, the compounds of thisinvention are believed to inhibit bacterial cell wall biosynthesisthereby inhibiting the growth of the bacteria or causing lysis of thebacteria. Accordingly, in another of its method aspects, this inventionprovides a method of inhibiting the growth of bacteria, the methodcomprising contacting bacteria with a growth-inhibiting amount of acompound of formula I, or a pharmaceutically-acceptable salt thereof,including any of the particular embodiments discussed herein.

Additionally, in yet another of its method aspects, this inventionprovides a method of inhibiting bacterial cell wall biosynthesis, themethod comprising contacting bacteria with a cell wallbiosynthesis-inhibiting amount of a compound of formula I, or apharmaceutically-acceptable salt thereof, including any of theparticular embodiments discussed herein.

This invention is also directed to processes for preparing compounds offormula I or a salt thereof. Accordingly, in another of its methodaspects, this invention provides a process for preparing a compound offormula I, or a salt thereof; the process comprising reacting a compoundof formula 1 or a salt, activated derivative, or protected derivativethereof, with a compound of formula 2 or a salt, activated derivative,or protected derivative thereof; and a compound of formula 3 or a salt,activated derivative, or protected derivative thereof, to form thecompound of formula I, wherein the compounds of formula 1, 2 and 3 areas defined herein.

In one embodiment, the above process further comprise the step offorming a pharmaceutically-acceptable salt of a compound of formula I.This invention is also directed to the product prepared by any of theseprocesses described herein.

This invention is also directed to a compound of formula I, or apharmaceutically acceptable salt thereof, for use in therapy.Additionally, this invention is directed to the use of a compound offormula I, or a pharmaceutically-acceptable salt thereof, for themanufacture of a medicament, including a medicament for treating abacterial infection in a mammal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows representative examples of cross-linkedglycopeptide—cephalosporin antibiotics in accordance with selectedembodiments of the invention.

FIG. 2 shows a representative process for preparing cephalosporinintermediates that are useful as intermediates for the compounds of theinvention.

FIG. 3 shows a representative process for preparing cross-linkedglycopeptide—cephalosporin antibiotics of the invention.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides novel glycopeptide—cephalosporin compounds offormula I, or pharmaceutically acceptable salts thereof. These compoundshave multiple chiral centers and, in this regard, the compounds areintended to have the stereochemistry shown. In particular, theglycopeptide portion of the compound is intended to have thestereochemistry of the corresponding naturally-occurring glycopeptide(i.e., vancomycin, chloroorienticin A and the like). The cephalosporinportion of the molecule is intended to have the stereochemistry of knowncephalosporin compounds. However, it will be understood by those skilledin the art that minor amounts of isomers having a differentstereochemistry from that shown may be present in the compositions ofthis invention provided that the utility of the composition as a wholeis not precluded by the presence of such isomers.

Additionally, the linking portion of the compounds of this invention maycontain one or more chiral centers. Typically, this portion of themolecule will be prepared as a racemic mixture. If desired, however,pure stereoisomers (i.e., individual enantiomers or diastereomers) maybe used or a stereoisomer-enriched mixture can be employed. All suchstereoisomers and enriched mixtures are included within the scope ofthis invention.

In addition, compounds of this invention contain several acidic groups(i.e., carboxylic acid groups) and several basic groups (i.e., primaryand secondary amine groups) and therefore, the compounds of formula Ican exist in various salt forms. All such salt forms are included withinthe scope of this invention. Also, since the compounds of formula Icontain a pyridinium ring, an anionic counterion for the pyridiniumgroup may optionally be present including, but not limited to, halides,such as chloride; carboxylates, such as acetate; and the like.

DEFINITIONS

The following terms, as used herein, have the following meanings, unlessotherwise indicated:

The term “alkyl” means a monovalent saturated hydrocarbon group whichmay be linear or branched. Unless otherwise defined, such alkyl groupstypically contain from 1 to 10 carbon atoms. Representative alkyl groupsinclude, by way of example, methyl, ethyl, n-propyl, isopropyl, n-butyl,sec-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl,n-nonyl, n-decyl and the like.

The term “alkylene” means a divalent saturated hydrocarbon group whichmay be linear or branched. Unless otherwise defined, such alkylenegroups typically contain from 1 to 10 carbon atoms. Representativealkylene groups include, by way of example, methylene, ethane-1,2-diyl(“ethylene”), propane-1,2-diyl, propane-1,3-diyl, butane-1,4-diyl,pentane-1,5-diyl and the like.

The term “alkenyl” means a monovalent unsaturated hydrocarbon groupwhich may be linear or branched and which has at least one, andtypically 1, 2 or 3, carbon-carbon double bonds. Unless otherwisedefined, such alkenyl groups typically contain from 2 to 10 carbonatoms. Representative alkenyl groups include, by way of example,ethenyl, n-propenyl, isopropenyl, n-but-2-enyl, n-hex-3-enyl and thelike.

The term “alkynyl” means a monovalent unsaturated hydrocarbon groupwhich may be linear or branched and which has at least one, andtypically 1, 2 or 3, carbon-carbon triple bonds. Unless otherwisedefined, such alkynyl groups typically contain from 2 to 10 carbonatoms. Representative alkynyl groups include, by way of example,ethynyl, n-propynyl, n-but-2-ynyl, n-hex-3-ynyl and the like.

The term “aryl” means a monovalent aromatic hydrocarbon having a singlering (i.e., phenyl) or fused rings (i.e., naphthalene). Unless otherwisedefined, such aryl groups typically contain from 6 to 10 carbon ringatoms. Representative aryl groups include, by way of example, phenyl andnaphthalene-1-yl, naphthalene-2-yl, and the like.

The term “arylene” means a divalent aromatic hydrocarbon having a singlering (i.e., phenylene) or fused rings (i.e., naphthalenediyl). Unlessotherwise defined, such arylene groups typically contain from 6 to 10carbon ring atoms. Representative arylene groups include, by way ofexample, 1,2-phenylene, 1,3-phenylene, 1,4-phenylene,naphthalene-1,5-diyl, naphthalene-2,7-diyl, and the like.

The term “cycloalkyl” means a monovalent saturated carbocyclichydrocarbon group. Unless otherwise defined, such cycloalkyl groupstypically contain from 3 to 10 carbon atoms. Representative cycloalkylgroups include, by way of example, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl and the like.

The term “cycloalkylene” means a divalent saturated carbocyclichydrocarbon group. Unless otherwise defined, such cycloalkylene groupstypically contain from 3 to 10 carbon atoms. Representativecycloalkylene groups include, by way of example, cyclopropane-1,2-diyl,cyclobutyl-1,2-diyl, cyclobutyl-1,3-diyl, cyclopentyl-1,2-diyl,cyclopentyl-1,3-diyl, cyclohexyl-1,2-diyl, cyclohexyl-1,3-diyl,cyclohexyl-1,4-diyl, and the like.

The term “halo” means fluoro, chloro, bromo and iodo.

The term “heteroaryl” means a monovalent aromatic group having a singlering or two fused rings and containing in the ring at least oneheteroatom (typically 1 to 3 heteroatoms) selected from nitrogen, oxygenor sulfur. Unless otherwise defined, such heteroaryl groups typicallycontain from 5 to 10 total ring atoms. Representative heteroaryl groupsinclude, by way of example, monovalent species of pyrrole, imidazole,thiazole, oxazole, furan, thiophene, triazole, pyrazole, isoxazole,isothiazole, pyridine, pyrazine, pyridazine, pyrimidine, triazine,indole, benzofuran, benzothiophene, benzimidazole, benzthiazole,quinoline, isoquinoline, quinazoline, quinoxaline and the like, wherethe point of attachment is at any available carbon or nitrogen ringatom.

The term “heteroarylene” means a divalent aromatic group having a singlering or two fused rings and containing at least one heteroatom(typically 1 to 3 heteroatoms) selected from nitrogen, oxygen or sulfurin the ring. Unless otherwise defined, such heteroarylene groupstypically contain from 5 to 10 total ring atoms. Representativeheteroarylene groups include, by way of example, divalent species ofpyrrole, imidazole, thiazole, oxazole, furan thiophene, triazole,pyrazole, isoxazole, isothiazole, pyridine, pyrazine, pyridazine,pyrimidine, triazine, indole, benzofuran, benzothiophene, benzimidazole,benzthiazole, quinoline, isoquinoline, quinazoline, quinoxaline and thelike, where the point of attachment is at any available carbon ornitrogen ring atom.

The term “heterocyclyl” or “heterocyclic” means a monovalent or divalentsaturated or unsaturated (non-aromatic) group having a single ring ormultiple condensed rings and containing in the ring at least oneheteroatom (typically 1 to 3 heteroatoms) selected from nitrogen, oxygenor sulfur. Unless otherwise defined, such heterocyclic groups typicallycontain from 2 to 9 total ring atoms. Representative heterocyclic groupsinclude, by way of example, monovalent species of pyrrolidine,imidazolidine, pyrazolidine, piperidine, 1,4-dioxane, morpholine,thiomorpholine, piperazine, 3-pyrroline and the like, where the point ofattachment is at any available carbon or nitrogen ring atom.

The term “cephalosporin” is used herein in its art-recognized manner torefer to a β-lactam ring system having the following general formula andnumbering system:

where R^(x) and R^(y) represent the remaining portion of thecephalosporin.

The term “glycopeptide antibiotic” or “glycopeptide” is used herein inits art-recognized manner to refer to the class of antibiotics known asglycopeptides or dalbahpeptides. See, for example, R. Nagarajan,“Glycopeptide Antibiotics”, Marcel Dekker, Inc. (1994) and referencescited therein. Representative glycopeptides include vancomycin, A82846A(eremomycin), A82846B (chloroorienticin A), A82846C, PA-42867-A(orienticin A), PA-42867-C, PA-42867-D and the like.

The term “vancomycin” is used herein in its art-recognized manner torefer to the glycopeptide antibiotic known as vancomycin. Whenvancomycin is employed in the compounds of the present invention, thepoint of attachment for the linking moiety is amino acid 7 (AA-&) atposition C-29. This position is also sometimes referred to as the “7d”or the “resorcinol” position of vancomycin.

The term “cross-linked glycopeptide—cephalosporin antibiotics” meanscovalent conjugation of a glycopeptide component to a cephalosporincomponent.

The term “pharmaceutically-acceptable salt” means a salt which isacceptable for administration to a patient, such as a mammal (e.g.,salts having acceptable mammalian safety for a given dosage regime).Such salts can be derived from pharmaceutically-acceptable inorganic ororganic bases and from pharmaceutically-acceptable inorganic or organicacids. Salts derived from pharmaceutically-acceptable inorganic basesinclude aluminum, ammonium, calcium, copper, ferric, ferrous, lithium,magnesium, manganic, manganous, potassium, sodium, zinc and the like.Particularly preferred are ammonium, calcium, magnesium, potassium andsodium salts. Salts derived from pharmaceutically-acceptable organicbases include salts of primary, secondary and tertiary amines, includingsubstituted amines, cyclic amines, naturally-occurring amines and thelike, such as arginine, betaine, caffeine, choline,N,N′-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol,2-dimethylaminoethanol, ethanolamine, ethylenediamine,N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine,hydrabamine, isopropylamine, lysine, methylglucamine, morpholine,piperazine, piperadine, polyamine resins, procaine, purines,theobromine, triethylamine, trimethylamine, tripropylamine, tromethamineand the like. Salts derived from pharmaceutically-acceptable acidsinclude acetic, ascorbic, benzenesulfonic, benzoic, camphosulfonic,citric, ethanesulfonic, fumaric, gluconic, glucoronic, glutamic,hippuric, hydrobromic, hydrochloric, isethionic, lactic, lactobionic,maleic, malic, mandelic, methanesulfonic, mucic, naphthalenesulfonic,nicotinic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric,tartaric, p-toluenesulfonic and the like. Particularly preferred arecitric, hydrobromic, hydrochloric, maleic, phosphoric, sulfuric andtartaric acids.

The term “salt thereof” means a compound formed when the hydrogen of anacid is replaced by a cation, such as a metal cation or an organiccation and the like (e.g., an NH₄ ⁺ cation and the like). Preferably,the salt is a pharmaceutically-acceptable salt, although this is notrequired for salts of intermediate compounds which are not intended foradministration to a patient.

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a patient in need of treatment.

The term “treating” or “treatment” as used herein means the treating ortreatment of a disease or medical condition (such as a bacterialinfection) in a patient, such as a mammal (particularly a human or acompanion animal) which includes:

(a) preventing the disease or medical condition from occurring, i.e.,prophylactic treatment of a patient;

(b) ameliorating the disease or medical condition, i.e., eliminating orcausing regression of the disease or medical condition in a patient;

(c) suppressing the disease or medical condition, i.e., slowing orarresting the development of the disease or medical condition in apatient; or

(d) alleviating the symptoms of the disease or medical condition in apatient.

The term “growth-inhibiting amount” means an amount sufficient toinhibit the growth or reproduction of a microorganism or sufficient tocause death or lysis of the microorganism including Gram-positivebacteria.

The term “cell wall biosynthesis-inhibiting amount” means an amountsufficient to inhibit cell wall biosynthesis in a microorganismincluding Gram-positive bacteria.

The term “leaving group” means a functional group or atom which can bedisplaced by another functional group or atom in a substitutionreaction, such as a nucleophilic substitution reaction. By way ofexample, representative leaving groups include chloro, bromo and iodogroups; and sulfonic ester groups, such as mesylate, tosylate,brosylate, nosylate and the like; activated ester groups, such as suchas 7-azabenzotriazole-1-oxy and the like; acyloxy groups, such asacetoxy, trifluoroacetoxy and the like.

The term “protected derivatives thereof” means a derivative of thespecified compound in which one or more functional groups of thecompound are protected from undesired reactions with a protecting orblocking group. Functional groups which may be protected include, by wayof example, carboxylic acid groups, amino groups, hydroxyl groups, thiolgroups, carbonyl groups and the like. Representative protecting groupsfor carboxylic acids include esters (such as a p-methoxybenzyl ester),amides and hydrazides; for amino groups, carbamates (such astert-butoxycarbonyl) and amides; for hydroxyl groups, ethers and esters;for thiol groups, thioethers and thioesters; for carbonyl groups,acetals and ketals; and the like. Such protecting groups are well-knownto those skilled in the art and are described, for example, in T. W.Greene and G. M. Wuts, Protecting Groups in Organic Synthesis, ThirdEdition, Wiley, N.Y., 1999, and references cited therein.

The term “amino-protecting group” means a protecting group suitable forpreventing undesired reactions at an amino group. Representativeamino-protecting groups include, but are not limited to,tert-butoxycarbonyl (BOC), trityl (Tr), benzyloxycarbonyl (Cbz),9-fluorenylmethoxycarbonyl (Fmoc), formyl, trimethylsilyl (TMS),tert-butyldimethylsilyl (TBS), and the like.

The term “carboxy-protecting group” means a protecting group suitablefor preventing undesired reactions at a carboxy group (i.e., —COOH).Representative carboxy-protecting groups include, but are not limitedto, esters, such as methyl, ethyl, tert-butyl, benzyl (Bn),p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm), trimethylsilyl (TMS),tert-butyldimethylsilyl (TBS), diphenylmethyl (benzhydryl, DPM) and thelike.

An “activated derivative”, with respect to a carboxylic acid orprotected derivative thereof, or such an acid or derivative in“activated form”, means the product, typically a reactive ester,resulting from reaction of the carboxylic acid or derivative with anactivating (coupling) agent, such as, for example,1-hydroxybenzotriazole (HOBT), 1-hydroxy-7-azabenzotriazole (HOAT), orothers described herein or otherwise known in the art.

A “side chain of a naturally occurring amino acid” means the group R inthe formula HOOC—CHR—NH₂, where this formula represents an amino acidselected from alanine, arginine, asparagine, aspartic acid, cysteine,glutamine, glutamic acid, glycine, histidine, isoleucine, leucine,lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine and valine; including the group selected fromalanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, isoleucine, leucine, lysine, methionine, serine,threonine and valine.

REPRESENTATIVE EMBODIMENTS

The following substituents and values are intended to providerepresentative examples and embodiments of various aspects of thisinvention. These representative values are intended to further definesuch aspects and embodiments and are not intended to exclude otherembodiments or limit the scope of this invention. In this regard, therepresentation that a particular value or substituent is preferred isnot intended in any way to exclude other values or substituents fromthis invention unless specifically indicated.

In compounds of formula I, each heteroaryl or heterocyclic group, whenpresent in R″, preferably has 5 or 6 total ring atoms; and each arylgroup, when present, preferably has 6 total ring atoms. The group R″ ispreferably C₁₋₁₂ alkylene, and is preferably linear.

In a specific embodiment, R¹ is hydrogen or C₁₋₄ alkyl, such as methylor ethyl. In another embodiment, R¹ is hydrogen.

In another specific embodiment, R² is hydrogen or C₁₋₄ alkyl, such asmethyl or ethyl. In another embodiment, R² is hydrogen.

Each R³ group, when present, is preferably independently selected fromC₁₋₄ alkyl, C₁₋₄ alkoxy, fluoro, and chloro. In one embodiment, n is 1or 2, and each R³ is independently selected from methyl, methoxy,fluoro, and chloro. In another embodiment, n is zero, such that no R³group is present.

The pyridinium ring in formula I is typically meta or para substituted,more generally para substituted.

In one embodiment, R⁸ is hydrogen. In another embodiment, R⁸ is a groupof the formula:

In a specific embodiment, R⁹ is hydrogen, C₁₋₄ alkyl and C₃₋₅cycloalkyl, where the alkyl group is optionally substituted with —COOHor 1 to 3 fluoro substituents; including hydrogen, methyl, ethyl,2-fluoroethyl, 2-carboxyprop-2-yl and cyclopentyl.

In one embodiment, W is CCl. In another embodiment, W is N.

Specific embodiments of other variables of formula I include,independent of each other, where X¹ and X² are both chloro; where R⁴ andR⁵ are OH and hydrogen, respectively; where R⁶ and R⁷ are hydrogen andmethyl, respectively.

In one embodiment, R^(a) is —Y—R″—, where R″ is C₁₋₆ alkylene, C₂₋₆alkenylene or C₂₋₆ alkynylene, and Y is selected from a direct bond,NR′, ether, sulfide, carbonyl, NR′C(O), and C(O)NR′, where R′ ishydrogen or methyl. In specific embodiments, in the group R^(a), Y is adirect bond, and R″ is C₁₋₆ alkylene, including C₁₋₄ alkylene, e.g.methylene.

In specific embodiments, x and y are independently selected from 0and 1. Accordingly, specific embodiments include compounds in which x+y=0, compounds in which x+y=1 (i.e., x is 1 and y is 0; or x is 0 and yis 1), and compounds in which x+y=2. Additionally, specific embodimentsinclude compounds in which the “linker” structure, represented by—R^(a)—[NR^(b)—C(O)—R^(c)]_(x)—[C(O)—NR^(d)—R^(e)]_(y)—NR²— in formulaI, is no more than about 40 atoms in length, and preferably no more thanabout 30 atoms in length (measured using the shorest number ofconsecutive atoms in the linker).

In selected embodiments, where x is not 0, and is preferably 1, theR^(b) is hydrogen, C₁₋₄ alkyl or C₂₋₄ alkenyl.

In one embodiment, R^(c) is —Y′—R″—Y′—, where each Y′ is independentlyselected from a direct bond, O (ether), and NR′, where R′ is hydrogen ormethyl, and R″ is selected from the group consisting of C₁₋₁₂ alkylene,C₂₋₁₂ alkenylene and C₂₋₁₂ alkynylene. Preferably, in the group R^(c),Y′ is a direct bond, and R″ is C₁₋₁₂ alkylene. More preferably, in thegroup R^(c), R″ is C₂₋₆ alkylene.

In other selected embodiments, where y is not 0, and is preferably 1,the variable R^(d) is hydrogen, C₁₋₄ alkyl or C₂₋₄ alkenyl, preferablyhydrogen or methyl, and more preferably hydrogen.

In one embodiment, R^(b) and R^(d) are independently hydrogen or methyl.

In one embodiment, R^(e) is selected from C₁₋₁₂ alkylene, C₂₋₁₂alkenylene and C₂₋₁₂ alkynylene; preferably, from C₁₋₆ alkylene, C₂₋₆alkenylene and C₂₋₆ alkynylene; and more preferably, from C₁₋₄ alkylene.

One exemplary class of compounds of formula I is that in which: x is 0or 1; y is 0 or 1; R^(a) is methylene; R^(b) (when x is 1) is hydrogen,methyl, or ethyl; R^(c) (when x is 1) is C₂₋₁₂ alkylene, e.g. n-butylene(—(CH₂)₄—) or n-decylene (—(CH₂)₁₀—), which may be substituted with—COOH; R^(d) (when y is 1) is hydrogen; and R^(e) (when y is 1) isethylene (—CH₂CH₂—).

In one embodiment, the compounds of this invention are those of formulaII. In formula II, a specific embodiment for W is CCl.

Specific embodiments for R⁹ are hydrogen, C₁₋₄ alkyl and C₃₋₅cycloalkyl, where the alkyl group is optionally substituted with —COOHor 1 to 3 fluoro substituents; including hydrogen, methyl, ethyl,2-fluoroethyl, 2-carboxyprop-2-yl and cyclopentyl.

Specific embodiments for R¹⁰ are hydrogen or methyl.

A specific embodiment for R¹¹ is C₁₋₁₀ alkylene, including C₁₋₆alkylene; such as—CH₂—, —(CH₂)₂—, —(CH₂)₃—, —(CH₂)₄—, —(CH₂)₅—and —(CH₂)₆—.

Specific embodiments for R¹² are hydrogen, C₁₋₄ alkyl or C₂₋₄ alkenyl;including hydrogen or methyl.

A specfic embodiment of a compound of formula II, is a compound whereinW is CCl; R⁹ is methyl; R¹⁰ is hydrogen; R¹¹ is —(CH₂)₄—; R¹² ishydrogen; and the pyridinium ring is para substituted.

As for formula I above, the pyridinium ring in formula II is typicallymeta or para substituted, more generally para substituted.

Specific embodiments of compounds of this invention include compounds offormula II, or pharmaceutically acceptable salts thereof, wherein thesubstituents are as defined in Table I:

TABLE I Pyridinium Cmpd. No. R⁹ R¹⁰ R¹¹ R¹² W Substitution Ia —CH₃ —H—(CH₂)₁₀— —H CCl para Ib —CH₃ —H —(CH₂)₄— —H CCl para Ic —CH₃ —H—(CH₂)₄— —H CCl meta Id —CH₃ —H —CH₂— —H CCl para Ie —CH₃ —H —CH₂— —HCCl meta If —CH₃ —H —(CH₂)₂— —H CCl para Ig —CH₃ —H —(CH₂)₂— —H CCl metaIh —CH₃ —H —(CH₂)₃— —H CCl para Ii —CH₃ —H —(CH₂)₃— —H CCl meta Ij —CH₃—H —(CH₂)₅— —H CCl para Ik —CH₃ —H —(CH₂)₅— —H CCl meta Il —CH₃ —H—(CH₂)₆— —H CCl para Im —CH₃ —H —(CH₂)₆— —H CCl meta In —CH₃ —H —(CH₂)₇——H CCl para Io —CH₃ —H —(CH₂)₇— —H CCl meta Ip —CH₃ —H —(CH₂)₈— —H CClpara Iq —CH₃ —H —(CH₂)₈— —H CCl meta Ir —CH₃ —H —(CH₂)₉— —H CCl para Is—CH₃ —H —(CH₂)₉— —H CCl meta It —CH₃ —H —(CH₂)₁₀— —H CCl meta Iu —CH₃ —H—(CH₂)₁₁— —H CCl para Iv —CH₃ —H —(CH₂)₁₁— —H CCl meta Iw —CH₃ —H—(CH₂)₁₂— —H CCl para Ix —CH₃ —H —(CH₂)₁₂— —H CCl meta Iy —CH₂CH₃ —H—(CH₂)₄— —H CCl para Iz —CH₂CH₃ —H —(CH₂)₄— —H CCl meta Iaa —OH —H—(CH₂)₄— —H CCl para Iab —OH —H —(CH₂)₄— —H CCl meta Iac —C(CH₃)₂COOH —H—(CH₂)₄— —H CCl para Iad —C(CH₃)₂COOH —H —(CH₂)₄— —H CCl meta Iae—CH₂CH₂F —H —(CH₂)₄— —H CCl para Iaf —CH₂CH₂F —H —(CH₂)₄— —H CCl metaIag -cyclopentyl —H —(CH₂)₄— —H CCl meta Iah -cyclopentyl —H —(CH₂)₄— —HCCl para Iai —CH₃ —CH₃ —(CH₂)₄— —H CCl para Iaj —CH₃ —CH₃ —(CH₂)₄— —HCCl meta Iak —CH₃ —H —(CH₂)₄— —CH₃ CCl para Ial —CH₃ —H —(CH₂)₄— —CH₃CCl meta Iam —CH₃ —H —(CH₂)₄— —H N para Ian —CH₃ —H —(CH₂)₄— —H N metaIao —CH₂CH₃ —H —(CH₂)₄— —H N para Iap —CH₂CH₃ —H —(CH₂)₄— —H N meta Iaq—OH —H —(CH₂)₄— —H N para Iar —OH —H —(CH₂)₄— —H N meta Ias —C(CH₃)₂COOH—H —(CH₂)₄— —H N para Iat —C(CH₃)₂COOH —H —(CH₂)₄— —H N meta Iau—CH₂CH₂F —H —(CH₂)₄— —H N para Iav —CH₂CH₂F —H —(CH₂)₄— —H N meta Iaw-cyclopentyl —H —(CH₂)₄— —H N meta Iax -cyclopentyl —H —(CH₂)₄— —H Npara Iay —CH₃ —CH₃ —(CH₂)₄— —H N para Iaz —CH₃ —CH₃ —(CH₂)₄— —H N metaIba —CH₃ —H —(CH₂)₄— —CH₃ N para Ibb —CH₃ —H —(CH₂)₄— —CH₃ N meta

While not intending to be limited by theory, the compounds of formulas Iare believed to inhibit bacterial cell wall biosynthesis, therebyinhibiting the growth of the bacteria or causing lysis of the bacteria.Accordingly, compound of formula I are useful as antibiotics.

Among other properties, compounds of the invention have been found topossess surprising and unexpected potency against Gram-positivebacteria, including methicillin-resistant Staphylococci aureus (MRSA),as described further herein below.

General Synthetic Procedures

The cross-linked glycopeptide—cephalosporin compounds of this inventioncan be prepared from readily available starting materials, such as theintermediate compounds 1–3 described herein. It will be appreciated thatwhere typical or preferred process conditions (i.e., reactiontemperatures, times, mole ratios of reactants, solvents, pressures,etc.) are given, other process conditions can also be used, asdetermined by one skilled in the art, unless otherwise stated. Optimumreaction conditions may vary with the particular reactants or solventused, but such conditions can be readily determined by one skilled inthe art by routine optimization procedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary or desired to preventcertain functional groups from undergoing undesired reactions. Suitableprotecting groups for a particular functional group, as well as suitableconditions for protection and deprotection of such functional groups,are well known in the art. Protecting groups other than thoseillustrated in the procedures described herein may be used, if desired.For example, numerous protecting groups, and means for theirintroduction and removal, are described in T. W. Greene and G. M. Wuts,Protecting Groups in Organic Synthesis, Third Edition, Wiley, N.Y.,1999, and references cited therein.

In one method of synthesis, the compounds of formula I are prepared byreacting a compound of formula 1:

where R¹–R⁸, R^(d), R^(e) X¹ and X² are as defined herein, or a salt, oran activated and/or protected derivative thereof, with a compound offormula 2:

where W, R³, R⁹, R^(a-e), and n are as defined herein, or a salt orcarboxy-protected derivative thereof; in the presence of a compound offormula 3:HOOC—R^(c)—COOH  3or a salt, activated derivative, or protected derivative thereof,wherein R^(c) is as defined herein; to form the compound of formula I,or a salt or protected derivative thereof. Preferred embodiments of thevariables in 1, 2 and 3 are as described herein.

In preparing compounds of formula II, variables in structures 1, 2,and/or 3 are defined as follows: n is 0; R^(a) is CH₂; R^(b) ishydrogen, C₁₋₄ alkyl or C₂₋₄ alkenyl (as defined for R¹⁰ above); R^(c)is C₁₋₁₂ alkylene or C₂₋₁₂ alkenylene (as defined for R¹¹ above); R²,R⁵, and R⁶ are hydrogen; R⁷ and R⁹ are CH₃; R⁴ is OH; and X¹ and X² arechloro.

Typically, for preparing compounds of formula I in which x=y=1, a lactamintermediate 2, having a primary or secondary amino group(—R^(a)—NHR^(b)) as shown, is reacted with an excess of bifunctionallinking reagent 3, with the latter in activated form (see FIG. 3).Employing an excess of 3 (typically a 3- to 10-fold molar excess; e.g. a5-fold molar excess, as shown in Example 3) favors formation of themonoadduct of 2 and 3, rather than a bis-adduct of 3 with two moleculesof 2. Preferably, 3 is provided as an activated derivative, such as thebis-HOAT derivative, and the reaction is catalyzed with an amine such as2,4,6-collidine. The adduct is then reacted with about 0.5 to about 2.5equivalents, preferably about 1.5 equivalents, relative to originallactam 3, of glycopeptide 1, or a salt thereof, in an inert solvent,such as DMF, containing a catalyst such as 2,4,6-collidine. The couplingreactions are generally carried out at a temperature ranging from about−20° C. to about 25° C., preferably in an ice bath (about 0–4° C.), forabout 15 minutes to 3 hours, or until the reaction is substantiallycomplete.

Intermediates of formula 1 can in turn be prepared by Mannich reaction(aminoalkylation) of the phenolic A-ring on a vancomycin-typeglycopeptide, employing the desired diamine (HR²N—R^(e)—NHR^(d)), analdehyde (R¹CHO, preferably where R¹=H) and base, as described inExample 2. Glycopeptides for preparation of the intermediates of formula1 are either commercially available or can be prepared by fermentationof the appropriate glycopeptide-producing organism, followed byisolation of the glycopeptide from the resulting fermentation brothusing art recognized procedures and equipment.

The cephalosporin intermediate 2 is readily prepared from commerciallyavailable starting materials and reagents using conventional procedures.By way of example, an intermediate of formula 2 can be prepared as shownin FIG. 2 and described in Example 1. Briefly,2-amino-5-chloro-α-methoxyimino-4-thiazole acetic acid, shown at 6 inFIG. 2, was reacted with the amino cephalosporinic ester 7, catalyzedwith EDAC, forming an amide linkage. This product (8) was reacted withsodium iodide in acetone, followed by displacement of the primary iodidewith a protected aminoalkyl pyridine derivative. The pyridine derivativecontains optional substituent(s) R³, as shown in structure 2 above. Inthe preparation shown in FIG. 2, the compound 4-(N-tert-BOC amino)methylpyridine (9) is employed, such that R^(a) is methylene and R^(b) ishydrogen. This reaction gives intermediate (10) in protected form;deprotection with TFA/anisole gives intermediate 2a (intermediate 2where W is CCl, R⁹ is Me, n is 0, R^(a) is CH₂ and R^(b) is hydrogen).

For preparing compounds of formula I where x=0, an intermediate such asa compound of formula 4:

wherein R^(a), R³, R⁹, W and n are as defined herein; is condensed withan intermediate of formula 1. Intermediate 4 can be prepared by avaration of the procedure given in Example 1, where a pyridinederivative substituted with a protected carboxalkyl group (—R^(a)COOH)is used in place of the aminoalkyl substituted pyridine.

For preparing compounds of formula I where y=0, a glycopeptidederivative of formula 5:

where R¹–R⁸, R^(c), X¹ and X² are as defined herein, or a salt, or anactivated and/or protected derivative thereof (i.e., formula 1 where—NHR²—R^(e)—NHR^(d) is replaced with —NHR²—R^(c)—COOH) is condensed withan intermediate of formula 2. Intermediates of formula 5 may be preparedaccording to variation of the preparation shown in Example 2, where anamino acid (R²HN—R^(c)—COOH, in carboxy-protected form), rather than adiamine, is used in the Mannich reaction (see e.g. J. H. Short and C. W.Ours, J Heterocyc. Chem. 12(5):869–76, October 1975).

For preparing compounds of formula I where x>1, one or more amino acidsof formula HOOC—R^(c)—NHR^(b) can be added to the reactive amine(—R^(a)—NHR^(b)) of intermediate 2, prior to reaction with 3 and 1 asdescribed above. Similarly, for preparation of compounds of formula I inwhich y>1, one or more amino acids of formula HOOC—R^(e)—NHR^(d) can beadded to the reactive amine (—NHR²) of intermediate 1, prior to reactionwith the adduct of 2 and 3 as described above.

Preferred coupling reagents, or activating reagents, for use in thesereactions include benzotriazol-1-yloxy tripyrrolidinophosphoniumhexafluorophosphate (PyBOP), preferably used in the amount of about 0.5to about 1.5 equivalents, preferably about 0.9 to about 1.1 equivalents,in combination with about 0.5 to about 1.5 equivalents, preferably about0.9 to about 1.1 equivalents, of 1-hydroxybenzotriazole (HOBT) or1-hydroxy-7-azabenzotriazole (HOAT). Other suitable coupling reagentsinclude O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU); bis(2-oxo-3-oxazolidinyl)phosphinic chloride(BOP-Cl); diphenylphosphoryl azide (DPPA); diphenylphosphinic chloride;diphenyl chlorophosphate (DPCP) and HOAT;1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDAC);pentafluorophenyl diphenylphosphinate, and the like.

After the coupling reaction is complete, any protecting groups presentin the product are then removed using conventional procedures andreagents. For example, deprotection of N-trityl, N-BOC(N-tert-butoxycarbonyl) and/or COO-PMB (para-methoxybenzyl ester) can beeffected by treatment with excess trifluoroacetic acid and excessanisole or triethylsilane in an inert diluent, such as dichloromethaneor heptane, at ambient temperature for about 1 to about 12 hours, oruntil the reaction is complete. The deprotected product can be purifiedusing conventional procedures, such as column chromatography, HPLC,recrystallization and the like.

Various substituted pyridines for use in the above reactions, or forpreparing compounds with varying substitution at R^(a) and/or R³, asdisclosed herein, are either commercially available or can be preparedfrom commercially available starting materials and reagents usingconventional procedures. For example, various aminoalkyl-substitutedpyridines are commercially available, e.g. aminomethyl pyridines, whereR^(a) is methylene, and aminoethyl pyridines, where R^(a) is ethylene,or can be prepared using standard organic synthesis procedures.Representative substituted pyridine derivatives for use in this reactioninclude those in which R³ is selected from methyl, methoxy, thiomethoxy,carboxythiomethoxy, fluoro, chloro, phenyl, cyclopropyl, carboxylicacid, carboxamide, and combinations thereof. For preparation ofcompounds in which Y, which links R″ to the pyridinium ring, is selectedfrom NR′, O (ether), S (sulfide), carbonyl, NR′(CO), and (CO)NR′),starting pyridine compounds are commercially available or can beprepared by well known procedures. For example, 3-hydroxypyridine,4-hydroxypyridine, 3-aminopyridine, 4-aminopyridine, 4-mercaptopyridine,nicotinic acid and isonicotinic acid are commerically available fromAldrich Chemical Co, Milwaukee, Wis.

In preparing compounds in which Y′, in the linker group R^(c), isselected from O (ether) and NR′ (rather than a direct bond), linkingmoieties including R^(c) will include one or more carbamate or urealinkages, rather than amide linkages. Such linkages can be formed byconventional methods. For example, an amine (such as —NHR^(b) inintermediate 3 can be reacted with an isocyanate or a chloroformate toform, respectively, a urea or carbamate linkage.

Further details regarding specific reaction conditions and proceduresfor preparing representative compounds of this invention orintermediates thereto are described in the Examples set forth below.

Pharmaceutical Compositions

The cross-linked glycopeptide—cephalosporin compounds of this inventionare typically administered to a patient in the form of a pharmaceuticalcomposition. Accordingly, in one of its composition aspects, thisinvention is directed to a pharmaceutical composition comprising apharmaceutically-acceptable carrier or excipient and a therapeuticallyeffective amount of a compound of formula I or a pharmaceuticallyacceptable salt thereof.

Any conventional carrier or excipient may be used in the pharmaceuticalcompositions of this invention. The choice of a particular carrier orexcipient, or combinations of carriers or excipients, will depend on themode of administration being used to treat a particular patient or typeof bacterial infection. In this regard, the preparation of a suitablepharmaceutical composition for a particular mode of administration, suchas oral, topical, inhaled or parenteral administration, is well withinthe scope of those skilled in the pharmaceutical arts. Additionally, theingredients for such compositions are commercially-available from, forexample, Sigma, P.O. Box 14508, St. Louis, Mo. 63178. By way of furtherillustration, conventional formulation techniques are described inRemington's Pharmaceutical Sciences, Mace Publishing Co., Philadelphia,Pa. 17^(th) Ed. (1985) and “Modern Pharmaceutics,” Marcel Dekker, Inc.3^(rd) Ed. (G. S. Banker & C. T. Rhodes, Eds.).

The pharmaceutical compositions of this invention will typically containa therapeutically effective amount of a compound of formula I or apharmaceutically-acceptable salt thereof. Typically, such pharmaceuticalcompositions will contain from about 0.1 to about 90% by weight of theactive agent, and more generally from about 10 to about 30% of theactive agent.

Preferred pharmaceutical compositions of this invention are thosesuitable for parenteral administration, particularly intravenousadministration. Such pharmaceutical compositions typically comprise asterile, physiologically-acceptable aqueous solution containing atherapeutically effective amount of a compound of formula I or apharmaceutically-acceptable salt thereof.

Physiologically-acceptable aqueous carrier solutions suitable forintravenous administration of active agents are well-known in the art.Such aqueous solutions include, by way of example, 5% dextrose, Ringer'ssolutions (lactated Ringer's injection, lactated Ringer's plus 5%dextrose injection, acylated Ringer's injection), Normosol-M, Isolyte E,and the like.

Optionally, such aqueous solutions may contain a co-solvent, forexample, polyethylene glycol; a chelating agent, for example,ethylenediamine tetraacetic acid; a solubilizing agent, for example, acyclodextrin; an anti-oxidant, for example, sodium metabisulphite; andthe like.

If desired, the aqueous pharmaceutical compositions of this inventioncan be lyophilized and subsequently reconstituted with a suitablecarrier prior to administration. In a preferred embodiment, thepharmaceutical composition is a lyophilized composition comprising apharmaceutically-acceptable carrier and a therapeutically effectiveamount of a compound of formula I, or a pharmaceutically-acceptable saltthereof. Preferably, the carrier in this composition comprises sucrose,mannitol, dextrose, dextran, lactose or a combination thereof. Morepreferably, the carrier comprises sucrose, mannitol, or a combinationthereof.

In one embodiment, the pharmaceutical compositions of this inventioncontain a cyclodextrin. When used in the pharmaceutical compositions ofthis invention, the cyclodextrin is preferablyhydroxypropyl-β-cyclodextrin or sulfobutyl ether β-cyclodextrin. In suchformulations, the cyclodextrin will comprise about 1 to 25 weightpercent; preferably, about 2 to 10 weight percent of the formulation.Additionally, the weight ratio of cyclodextrin to active agent willtypically range from about 1:1 to about 10:1.

The pharmaceutical compositions of this invention are preferablypackaged in a unit dosage form. The term “unit dosage form” means aphysically discrete unit suitable for dosing a patient, i.e., each unitcontaining a predetermined quantity of active agent calculated toproduce the desired therapeutic effect either alone or in combinationwith one or more additional units. For example, such unit dosage formsmay be packaged in sterile, hermetically-sealed ampoules and the like.

The following formulations illustrate representative pharmaceuticalcompositions of the present invention:

Formulation Example A

A frozen solution suitable for preparing an injectable solution isprepared as follows:

Ingredients Amount Compound of the Invention 10 to 1000 mg Excipients(e.g., dextrose) 0 to 50 g Water for Injection Solution 10 to 100 mL

Representative Procedure: The excipients, if any, are dissolved in about80% of the water for injection and the active compound is added anddissolved. The pH is adjusted with 1 M sodium hydroxide to 3 to 4.5 andthe volume is then adjusted to 95% of the final volume with water forinjection. The pH is checked and adjusted, if necessary, and the volumeis adjusted to the final volume with water for injection. Theformulation is then sterile filtered through a 0.22 micron filter andplaced into a sterile vial under aseptic conditions. The vial is capped,labeled and stored frozen.

Formulation Example B

A lyophilized powder suitable for preparing an injectable solution isprepared as follows:

Ingredients Amount Compound of the Invention 10 to 1000 mg Excipients(e.g., mannitol and/or sucrose) 0 to 50 g Buffer Agent (e.g., citrate) 0to 500 mg Water for Injection 10 to 100 mL

Representative Procedure: The excipients and/or buffering agents, ifany, are dissolved in about 60% of the water for injection. The activecompound is added and dissolved and the pH is adjusted with 1 M sodiumhydroxide to 3 to 4.5 and the volume is adjusted to 95% of the finalvolume with water for injection. The pH is checked and adjusted, ifnecessary, and the volume is adjusted to the final volume with water forinjection. The formulation is then sterile filtered through a 0.22micron filter and placed into a sterile vial under aseptic conditions.The formulation is then freeze-dried using an appropriate lyophilizationcycle. The vial is capped (optionally under partial vacuum or drynitrogen), labeled and stored under refrigeration.

Formulation Example C

An injectable solution for intravenous administration to a patient isprepared from Formulation Example B above as follows:

Representative Procedure: The lyophilized powder of Formulation ExampleB (e.g., containing 10 to 1000 mg of active compound) is reconstitutedwith 20 mL of sterile water and the resulting solution is furtherdiluted with 80 mL of sterile saline in a 100 mL infusion bag. Thediluted solution is then administered to the patient intravenously over30 to 120 minutes.

Utility

The cross-linked glycopeptide—cephalosporin compounds of the inventionare useful as antibiotics. For example, the compounds of this inventionare useful for treating or preventing bacterial infections and otherbacteria-related medical conditions in mammals, including humans andtheir companion animals (i.e., dogs, cats, etc.), that are caused bymicroorganisms susceptible to the compounds of this invention.

Accordingly, in one of its method aspects, the invention provides amethod of treating a bacterial infection in a mammal, the methodcomprising administering to a mammal in need of such treatment, apharmaceutical composition comprising a pharmaceutically-acceptablecarrier and a therapeutically effective amount of a compound of formulaI, or a pharmaceutically-acceptable salt thereof.

By way of illustration, the compounds of this invention are particularlyuseful for treating or preventing infections caused by Gram-positivebacteria and related microorganisms. For example, the compounds of thisinvention are effective for treating or preventing infections caused bycertain Enterococcus spp.; Staphylococcus spp., including coagulasenegative staphylococci (CNS); Streptococcus spp.; Listeria spp.;Clostridium ssp.; Bacillus spp.; and the like. Examples of bacterialspecies effectively treated with the compounds of this inventioninclude, but are not limited to, methicillin-resistant Staphylococcusaureus (MRSA); methicillin-susceptible Staphylococcus aureus (MSSA);glycopeptide intermediate-susceptible Staphylococcus aureus (GISA);methicillin-resistant Staphylococcus epidermitis (MRSE);methicillin-sensitive Staphylococcus epidermitis (MSSE);vancomycin-sensitive Enterococcus faecalis (EFSVS); vancomycin-sensitiveEnterococcus faecium (EFMVS); penicillin-resistant Streptococcuspneumoniae (PRSP); Streptococcus pyogenes; and the like.

As shown in Table II of Example 6 below, compounds Ia–c were moreeffective than vancomycin, by a factor of 10 or more, againstmethicillin-sensitive Staphylococcus aureus and methicillin-resistantStaphylococcus aureus. Compound Ic was also significantly more activethan its des-chloro analog “des-Cl Ic,” although this compound was alsomore active than vancomycin against MSSA. In a “time-kill” assay, asdescribed in Example 7, a compound of formula I, i.e. compound lb, wasbactericidal against MRSA at a concentration of 1.0 μg/mL in 4 hours. Bycomparison, vancomycin was bactericidal against MRSA at a concentrationof 4 μg/mL in 24 hours. In an in vivo assay in neutropenic mice, asdescribed in Example 8, a compound of formula I, i.e. compound Ib, hadan ED₅₀ of less than 0.1 mg/kg, iv, compared to an ED₅₀ of 9 mg/kg, iv,for vancomycin.

In general, the compounds of the invention are preferred for treating orpreventing infections caused by strains of bacteria which aresusceptible to either glycopeptides or cephalosporins.

Representative types of infections or bacteria-related medicalconditions which can be treated or prevented with the compounds of thisinvention include, but are not limited to, skin and skin structureinfections, urinary tract infections, pneumonia, endocarditis,catheter-related blood stream infections, osteomyelitis, and the like.In treating such conditions, the patient may already be infected withthe microorganism to be treated or merely be susceptible to infection inwhich case the active agent is administered prophylactically.

The compounds of this invention are typically administered in atherapeutically effective amount by any acceptable route ofadministration. Preferably, the compounds are administered parenterally.The compounds may be administered in a single daily dose or in multipledoses per day. The treatment regimen may require administration overextended periods of time, for example, for several days or for one tosix weeks or longer. The amount of active agent administered per dose orthe total amount administered will typically be determined by thepatient's physician and will depend on such factors as the nature andseverity of the infection, the age and general health of the patient,the tolerance of the patient to the active agent, the microorganism(s)causing the infection, the route of administration and the like.

In general, suitable doses will range of from about 0.25 to about 10.0mg/kg/day of active agent, preferably from about 0.5 to about 2mg/kg/day. For an average 70 kg human, this would amount to about 15 toabout 700 mg per day of active agent, or preferably about 35 to about150 mg per day.

Additionally, the compounds of this invention are effective forinhibiting the growth of bacteria. In this embodiment, bacteria arecontacted either in vitro or in vivo with a growth-inhibiting amount ofa compound of formula I or pharmaceutically-acceptable salt thereof.Typically, a growth-inhibiting amount will range from about 0.008 μg/mLto about 50 μg/mL; preferably from about 0.008 μg/mL to about 25 μg/mL;and more preferably, from about 0.008 μg/mL to about 10 μg/mL.Inhibition of bacterial growth is typically evidenced by a decrease orlack of reproduction by the bacteria and/or by lysis of the bacteria,i.e., by a decrease in colony-forming units in a given volume (i.e., permL) over a given period of time (i.e., per hour) compared to untreatedbacteria.

The compounds of this invention are also effective for inhibiting cellwall biosynthesis in bacteria. In this embodiment, bacterial arecontacted either in vitro or in vivo with a cell wallbiosynthesis-inhibiting amount of a compound of formula I orpharmaceutically-acceptable salt thereof. Typically, a cell wallbiosynthesis-inhibiting amount will range from about 0.04 μg/mL to about50 μg/mL; preferably from about 0.04 μg/mL to about 25 μg/mL; and morepreferably, from about 0.04 μg/mL to about 10 μg/mL. Inhibition of cellwall biosynthesis in bacteria is typically evidenced by inhibition orlack of growth of the bacteria including lysis of the bacteria.

Additionally, compounds of this invention have been found to havesurprising and unexpectedly rapid cidality against certain bacteria,including methicillin-resistant Staphylococci aureus (MRSA) andmethicillin-resistant Staphylococci epidermitis (MRSE). Theseproperties, as well as the antibiotic utility of the compounds of thisinvention, can be demonstrated using various in vitro and in vivo assayswell-known to those skilled in the art. For example, representativeassays are described in further detail in the following Examples.

EXAMPLES

The following synthetic and biological examples are offered toillustrate this invention and are not to be construed in any way aslimiting the scope of this invention.

In the examples below, the following abbreviations have the followingmeanings unless otherwise indicated. Abbreviations not defined belowhave their generally accepted meaning.

BOC = tert-butoxycarbonyl CFU = colony-forming units DCM =dichloromethane DIPEA = diisopropylethylamine DMF =N,N-dimethylformamide DMSO = dimethyl sulfoxide EtOAc = ethyl acetateHOBT = 1-hydroxy benzotriazole HOAT = 1-hydroxy-7-azabenzotriazole HPLC= high performance liquid chromatography MIC = minimum inhibitoryconcentration MS = mass spectrometry PMB = p-methoxybenzyl PyBOP =benzotriazol-1-yloxytripyrrolidino-phosphonium hexafluorophosphate THF =tetrahydrofuran TLC = thin layer chromatography TFA = trifluoroaceticacid

All temperatures reported in the following examples are in degreesCelsius (° C.) unless otherwise indicated. Also, unless noted otherwise,reagents, starting materials and solvents were purchased from commercialsuppliers (such as Aldrich, Fluka, Sigma and the like) and were usedwithout further purification. Vancomycin hydrochloride semi-hydrate waspurchased from Alpharma, Inc., Fort Lee, N.J. 07024 (Alpharma AS, Oslo,Norway).

Reverse-phase HPLC was typically conducted using a Cl₁₈ column and (A)98% water, 2% acetonitrile, 0.1% TFA, with an increasing gradient (e.g.,0 to about 70%) of (B) 10% water, 90% acetonitrile, 0.1% TFA, unlessotherwise stated.

Example 1 Preparation of 2a:(7R)-7-[(Z)-2-(2-Amino-5-chlorothiazol-4-yl)-2-(methoxyimino)acetamido]-3-[(4-(aminomethyl)-1-pyridinio)methyl]-3-cephem-4-carboxylateBis-trifluoroacetic Acid Salt (see FIG. 2)

A. Preparation of 2-Amino-5-Chloro-α-(Methoxyimino)-4-ThiazoleaceticAcid (6)

To 500 mL of DMF were added 50.0 g (250 mmol) of2-amino-α-(methoxyimino)-4-thiazoleacetic acid and 35 g (260 mmol) ofN-chlorosuccinimide. The mixture was stirred at room temperatureovernight, after which time mass spectral analysis showed no morestarting material to be present. The light brown solution was usedwithout further purification.

B. Preparation of Cephalosporin Derivative (8)

To the solution of the acid 6 in DMF from Step (a) was added 101.5 g(250 mmol) of the aminocephalosporonic ester 7, 34 g (250 mmol). Themixture was cooled to 0° C., and 33.5 mL (250 mmol) of 2,4,6-collidinewas added. To this solution was added 53 g (275 mmol) of1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. After 2hours, the solution was precipitated into 3 L water and filtered. Thesolids were washed with water (2×1 L), saturated sodium bicarbonate (500mL) and water (4×500 mL) and dried under vacuum. The dried solids weretaken up in 500 mL of methylene chloride at room temperature, and thesolution was slowly stirred, forming a precipitate. The crystals werecollected by filtration, washed with methylene chloride until thewashings were no longer brown, and dried under vacuum to give the amide8 (74 g).

Analytical Data: MS m/z calc. 586.04, obs. 586.2 (M+1); ¹H NMR(DMSO-d₆): δ9.60 (d, 1H), 7.35 (m, 3H), 6.91 (d, 2H), 5.82 (m, 1H), 5.17(m, 3H), 4.56 (m, 2H), 3.84 (s, 3H) 3.76 (s, 3H), 3.62 (m, 2H).

C. Preparation of Cephalosporin Derivative (10)

Acetone (250 mL) was added to a mixture of 50 g (85 mmol) of thechloromethylcephalosporin ester 8 and 13 g (85 mmol) of sodium iodide,under nitrogen in the dark. After stirring for 30 minutes, 27 g (130mmol) of 4-(N-tert-butoxycarbonyl)aminomethyl pyridine (9) and 30 mL ofacetone were added. After stirring an additional 2 hours, 1.4 L of 0.1NHCl was added, producing a gummy precipitate. The solvent portion wasdecanted, and the gummy residue was treated with 800 mL of water to givea solid. The water was decanted, and the solid was dissolved in 1 L of4:1 ethyl acetate/ethanol. The solution was washed with 500 mL ofsaturated brine, dried over magnesium sulfate, and evaporated to drynessto give 70 g (79 mmol, 93%) of product 10, having a purity of of 78% asdetermined by HPLC (254 nm).

D. Preparation of Cephalosporin Derivative (2a)

The cephalosporin derivative, 10, was deprotected as follows. The crudeproduct (70 g, 79 mmol) was dissolved in 550 mL of methylene chlorideunder nitrogen, and 35 mL (320 mmol) of anisole were added, followed by150 mL of trifluoroacetic acid. After 2 hours, the mixture wasconcentrated under vacuum. The product precipitated on addition of 1 Lof diethyl ether. The solids were isolated by filtration, washed withether, stirred in 200 mL of water and filtered. The filtrate waslyophilized to dryness and purified by reverse-phase HPLC, yielding 30 g(approx. 50%) of compound 2a, as the bis-TFA salt, which was usedwithout further purification.

Example 2 Preparation of 1a: Formula I where R¹, R², R⁵, R⁶, R⁸═H;R⁴═OH; R⁷═Me; X¹, X2═Cl; R^(d)═H; and R^(e)═—CH₂CH₂—

Under nitrogen, vancomycin hydrochloride monohydrate (20 g, 13 mmol) wasdissolved in water (100 mL) and cooled in an ice bath. Ethylenediamine(7 mL, 100 mmol) was added, followed by 1N NaOH (50 mL, 50 mmol).Formaldehyde (1.3 mL 37% aqueous H₂CO, 17 mmol) was added and thereaction mixture was kept in the dark at 4° C. overnight. HPLC analysisof the reaction mixture showed 78% of the desired product 1a, plusunreacted vancomycin and a bis-addition product. The reaction mixturewas acidified at 4° C. and the product recovered and purified by HPLC.

Example 3 Preparation of Compound Ib (see FIG. 3)

Adipic acid (3 where R^(c)=n-butylene) bis-HOAT ester (3a, 6.5 mmol) wasdissolved in DMF (50 mL) and pyridinium lactam bis-trifluoroacetate 2a(1.0 g, 1.3 mmol), prepared as described in Example 1 above, was added.The solution was then cooled in an ice bath and 2,4,6-collidine (342 μL,2.6 mmol) was added, and the mixture was stirred in the ice bath for 15minutes, followed by quenching with 300 μL TFA (3.9 mmol). The reactionmixture was then poured into 400 mL of ethyl acetate, and theprecipitated solids were collected by centrifugation.

A solution of 3.86 g (1.95 mmol) 1a, prepared as described in Example 2,in DMF (40 mL) was added to the collected solid, and the resultingmixture was cooled in an ice bath, followed by addition of2,4,6-collidine (1.03 μL, 7.8 mmol). The mixture was stirred in the icebath for 20 minutes, then trifluoroacetic acid was added (800 μL, 10.4mmol). The mixture was then poured into acetonitrile (400 mL), and theprecipitated solid was purified by reverse-phase HPLC to give theproduct Ib (740 mg, 0.29 mmol, 22% yield).

Analytical Data: MS m/z 2171.8 (MH⁺).

Example 4 Preparation of Compounds Ia, Ic, and Id

These compounds were prepared according to the procedures described inExamples 1, 2 and 3, substituting the appropriate starting materials.

Analytical Data:

Compound Ia: MS m/z fragments 1127.7, 1681.6, 1824.8 (MH⁺);

Compound Ic: MS m/z 2171.5 (MH⁺); and

Compound des-Cl Ic: The des-chloro derivative of Compound Ic (i.e.,where the chloro atom in the thiadiazole ring is replaced with hydrogen)was prepared for comparison purposes: MS m/z 2137.6 (MH⁺).

Example 5 Determination of Aqueous Solubility

The aqueous solubility of compounds of the invention was determinedusing the following procedure. A 5 wt. % dextrose buffer solution at pH2.2 was prepared by adding 1 mL of 1 N hydrochloric acid (Aldrich) to 99mL of a 5 wt. % aqueous dextrose solution (Baxter). A 1 mg/mL stocksolution for calibration standards was then prepared by dissolving 1 mgof the test compound in 1 mL of DMSO. This solution was vortexed for 30seconds and then sonicated for 10 minutes. The stock solution was thendiluted with water to prepare calibration standards having the followingconcentrations: 50, 125, 250, 375 and 500 μg/mL.

Each test compound (30 mg) was weighed into a Millipore non-sterile,Ultrafree-MC 0.1 μm filter unit (Millipore UFC30VVOO) and a magneticstir bar was added to each unit. The 5 wt. % dextrose buffer solution(750 μL) was then added to each unit and these mixtures were vortexedfor 5 minutes. The filter units were then placed in an Eppendorf tuberack and the tube rack was placed on top of a magnetic stirrer. Eachunit was then titrated to pH 3 using 1 N NaOH (VWR) and the resultingsolutions centrifuged at 7000 rpms for 5 minutes. Each unit was thendiluted 200 fold with 5% dextrose buffer solution and the dilutedsamples were transferred into auto sampler vials for analysis.

The calibration standards and the test samples were analyzed byreverse-phase HPLC using the following conditions:

Column: Luna 150×4.6 mm; C18; 5μ

Mobile phase: A=5/95, B=95/5, both=MeCN/H₂O; 0.1% TFA

Method: 10 m Lido 100 (0–100% B in 6 min)

Injection volume: 20 μL

Wavelength: 214 nm

The solubility of each test sample was calculated by comparing the peakarea of the test sample to the calibration curve and multiplying by thedilution factor.

According to the above procedure, the solubility of compound Ib in 5%aqueous dextrose buffer at pH 3 was determined to be greater than 7mg/mL.

Example 6 Determination of Minimal Inhibitory Concentrations (MIC)

Minimal inhibitory concentration (MIC) assays were performed using thebroth microdilution method set forth in NCCLS guidelines (see, NCCLS.2000. Methods for Dilution Antimicrobial Susceptibility Tests forBacteria That Grow Aerobically; Approved Standard—Fifth Ed., Vol. 20,No. 2). Bacterial strains were obtained from the American Type TissueCulture Collection (ATCC), Stanford University Hospital (SU), KaiserPermanente Regional Laboratory in Berkeley (KPB), Massachusetts GeneralHospital (MGH), the Centers for Disease Control (CDC), the San FranciscoVeterans' Administration Hospital (SFVA) or the University of CaliforniaSan Francisco Hospital (UCSF). Vancomycin-resistant enterococci werephenotyped as Van A or Van B based on their sensitivity to teicoplanin.Some vancomycin-resistant enterococci genotyped as Van A, Van B, Van C1or Van C2 were also obtained from the Mayo Clinic.

In this assay, cryopreserved bacterial cultures of reference andclinical strains were streaked for isolation on appropriate agar medium(i.e., Trypticase Soy Agar, Trypticase Soy Agar with defibrinated sheeperthrocytes, Brain Heart Infusion Agar, Chocolate Agar). Followingincubation to allow formation of colonies, these plates were sealed withparafilm and stored refrigerated for up to two weeks. For preparation ofassay inocula and to ensure low variability, several colonies from abacterial isolate cultured on the agar plates were pricked with aninoculating loop and aseptically transferred to Mueller-Hinton Broth(supplemented with divalent cations to required levels based onmanufacturer's certification). The broth culture was grown overnight at35° C., diluted in fresh prewarmed broth and grown to log phase; this isequivalent to a 0.5 MacFarland standard or 1×10⁸ colony forming unitsper milliliter (CFU/mL). Not all cell suspensions, due to speciesvariability, contained 1×10⁸ CFU/mL when turbidity is equivalent to theMacFarland standard, therefore acceptable adjustments (based on NCCLSguidelines) were made in dilutions of different bacterial strains. Theinoculum was diluted such that 100 μL of this culture in Mueller-HintonBroth, supplemented Mueller-Hinton Broth, or Haemophilus test medium,when over layered onto a 2-fold serially diluted series of antibioticconcentrations also in 100 μL of corresponding medium, in a 96-wellmicrotiter plate resulted in a starting bacterial concentration of 5×10⁵CFU/mL. The plates were then incubated 18–24 hours at 35° C. The MIC wasread visually as the lowest concentration well with no bacterial growth.Bacterial growth is defined as more than three pinpoint colonies, abutton of precipitated cells larger than 2 mm in diameter, or obviousturbidity.

Strains routinely tested in the initial screen includedmethicillin-sensitive Staphylococcus aureus (MSSA),methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcusaureus producing penicillinase, methicillin-sensistive Staphylococcusepidermidis (MSSE), methicillin-resistant Staphylococcus epidermidis(MRSE), vancomycin-sensitive Enterococcus faecium (EFMVS),vancomycin-sensitive Enterococcus faecalis (EFSVS), vancomycin-resistantEnterococcus faecium also resistant to teicoplanin (EFMVR Van A),vancomycin-resistant Enterococcus faecium sensistive to teicoplanin(EFMVR Van B), vancomycin-resistant Enterococcus faecalis also resistantto teicoplanin (EFSVR Van A), vancomycin-resistant Enterococcus faecalissensitive to teicoplanin (EFSVR Van B), penicillin-sensitiveStreptococcus pneumoniae (PS SP) and penicillin-resistant Streptococcuspneumoniae (PSRP). Because of the inability of PSSP and PSRP to growwell in Mueller-Hinton broth, MICs with those strains were determinedusing either TS broth supplemented with defibrinated blood orHaemophilus test medium.

Test compounds having significant activity against the strains mentionedabove were then tested for MIC values in a larger panel of clinicalisolates including the species listed above as well as non-speciatedcoagulase negative Staphylococcus both sensitive and resistant tomethicillin (MS-CNS and MR-CNS). Additionally, these test compounds werealso assayed for MICs against gram-negative microorganisms, such asEscherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae,Enterobacter cloacae, Acinetobacter baumannii, Haemophilius influenzaeand Moraxella catarrhalis.

Table II shows MIC₉₀ data for compounds of this invention againstmethicillin-resistant S. aureus (MRSA) and methicillin-susceptible S.aureus (MSSA) as compared to the known glycopeptide antibiotic,vancomycin.

TABLE II Minimum Inhibitory Concentrations (MICs), in μg/mL Compound MIC(μg/mL) MRSA 33591 MSSA 13709 Ia 0.15 0.15 Ib <0.1 <0.1 Ic 0.1 <0.1des-C1 Ic 3.13 <0.1 Vancomycin 2.0 1.0

The data in Table II demonstate that compounds of this invention (i.e.,Ia, Ib and Ic) had surprising and unexpected antibacterial activityagainst MRSA 33591 compared to either a des-chloro analog or vancomycin;and that compounds of this invention had suprising and unexpectedantibacterial acitivity against MSSA 13709 compared to vancomycin.

Example 7 Time-Kill Assay

This time-kill assay is a method for measuring the rate of bactericidalactivity of a test compound. These procedures are similar to thosedescribed in V. Lorian, “Antibiotics in Laboratory Medicine”, FourthEdition, Williams and Wilkins (1996), pages 104–105. A rapid time-killis desirable to quickly prevent bacterial colonization and reduce hosttissue damage.

Bacterial inocula were prepared as described in Example 6 fordetermination of MIC. Bacteria were diluted in prewarmed media in shakeflasks and incubated with shaking (200 rpm, 35° C.). At 0, 1, 4, and 24hours samples were withdrawn from the flasks and bacteria wereenumerated by plate counting. Subsequent to the initial sampling, a testcompound to be assayed was added to the shake flask culture. Platecounts at these intervals previous to and following addition of thecompound were expressed graphically in a time-kill curve. Bactericidalactivity is defined as a greater than or equal to 3 log decrease(reduction greater than or equal to 99.9%) in bacterial cell numbers by24 hours.

In this assay, a compound of formula I, i.e. compound Ib, wasbactericidal against MRSA 33591 at a concentration of 1.0 μg/mL in 4hours. By comparison, vancomycin was bactericidal against MRSA 33591 ata concentration of 4 μg/mL in 24 hours.

Example 8 In vivo Efficacy Studies in Neutropenic Mice

Animals (male CD-1 mice, 20–30g) were acquired from Charles RiversLaboratories (Gilroy, Calif. and allowed access to food and water adlibitum. Neutropenia was induced via 200 mg/kg intraperitoneal (IP)injection of cyclophosphamide given four and two days prior to theinoculation of bacteria.

The organism used was either a susceptible or resistant strain ofclinically relevant gram positive pathogens, such asmethicillin-susceptible Staphylococcus aureus (MSSA 13709) andmethicillin-resistant Staphylococcus aureus (MRSA 33591). The bacterialinoculum concentration was ˜10⁶ CFU/mL. Animals were lightlyanesthetized with isoflurane and 50 mL of the bacterial inoculum wasinjected into the anterior thigh. One hour after the inoculation,animals were dosed intravenously with vehicle or the appropriate dose ofthe test compound. At 0 hours and 24 hours post-treatment, the animalswere euthanized (CO₂ asphyxiation) and the anterior and posterior thighcollected aseptically. The thigh was placed into 10 mL sterile salineand homogenized. Dilutions of the homogenate were plated onto tripticsoy agar plates which were incubated overnight. The number of bacterialcolonies on a given plate was multiplied by the dilution factor, dividedby the thigh weight (in grams) and expressed as log CFU/g. ED₅₀ (doserequired to produce 50% of the maximum reduction in thigh titre) wasestimated for each test compound.

In this assay, a compound of formula I, i.e. compound Ib, had an ED₅₀ ofless than 7 mg/kg, iv, compared to an ED₅₀ of 9 mg/kg, iv, forvancomycin.

While the present invention has been described with reference tospecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made an equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto. Additionally, to the extent permitted byapplicable patent laws and regulations, all publications, patents, andpatent documents cited herein are incorporated by reference herein intheir entirety to the same extent as if they had been individuallyincorporated by reference.

1. A compound of formula I:

or a pharmaceutically-acceptable salt thereof; wherein each of X¹ and X²is independently hydrogen or chloro; W is N or CCl; R¹ and R² areindependently selected from hydrogen and C₁₋₆ alkyl; each R³ isindependently selected from C₁₋₆ alkyl, OR, halo, —SR, —S(O)R, —S(O)₂R,and —S(O)₂OR, where each R is independently C₁₋₆ alkyl optionallysubstituted with COOH or 1 to 3 fluoro substituents; one of R⁴ and R⁵ ishydroxy and the other is hydrogen; R⁶ and R⁷ are independently hydrogenor methyl; R⁸ is hydrogen or a group of the formula:

R⁹ is selected from hydrogen, C₁₋₆ alkyl and C₃₋₆ cycloalkyl, wherealkyl and cycloalkyl are optionally substituted with —COOH or 1 to 3fluoro substituents; R^(a) is —Y—R″—, where R″ is selected from C₁₋₁₂alkylene, C₂₋₁₂ alkenylene, C₂₋₁₂ alkynylene, C₃₋₆ cycloalkylene, C₆₋₁₀arylene, C₂₋₉ heteroarylene, C₃₋₆ heterocycle and combinations thereof,and is optionally substituted with 1 or 2 groups selected from Z, whereeach Z is independently selected from the group consisting of —OR′,—SR′, —F, —Cl, —N(R′)₂, —OC(O)R′, —C(O)OR′, —NHC(O)R′, —C(O)N(R′)₂,—CF₃, —OCF₃, and side chains of naturally-occurring amino acids, whereeach R′ is independently hydrogen or C₁₋₄ alkyl; and R″ contains at most20 non-hydrogen atoms; and Y, which links R″ to the pyridinium ring at ameta or para position, is selected from a direct bond, NR′, O, S, C(O),NR′C(O), and C(O)NR′, precluding direct bonds between heteroatoms in Yand R″; each R^(b) and R^(d) is independently selected from hydrogen,C₁₋₆ alkyl, C₂₋₆ alkenyl and C₂₋₆ alkynyl; each R^(c) is independently adirect bond or —Y′—R″—Y′—, where each Y′ is independently selected froma direct bond, O and NR′, precluding direct bonds between heteroatoms inY′ and R″; each R^(e) is independently selected from the group definedby R″; n is an integer from 0 to 3; x is an integer from 0 to 2; and yis an integer from 0 to
 2. 2. The compound of claim 1, wherein R⁹ ishydrogen, C₁₋₄ alkyl or C₃₋₅ cycloalkyl, where the alkyl group isoptionally substituted with —COOH or 1 to 3 fluoro substituents.
 3. Thecompound of claim 2, wherein R⁹ is hydrogen, methyl, ethyl,2-fluoroethyl, 2-carboxyprop-2-yl or cyclopentyl.
 4. The compound ofclaim 1, wherein W is CCl.
 5. The compound of claim 1, wherein W is N.6. The compound of claim 1, wherein each R³ is independently selectedfrom C₁₋₄ alkyl, C₁₋₄ alkoxy, fluoro and chloro.
 7. The compound ofclaim 1, wherein n is
 0. 8. The compound of claim 1, wherein x is 0 andy is
 1. 9. The compound of claim 1, wherein x is 1 and y is
 0. 10. Thecompound of claim 1, wherein x is 1 and y is
 1. 11. The compound ofclaim 10, wherein R^(a) is —Y—R″—, where R″ is C₁₋₆ alkylene; and Y is adirect bond.
 12. The compound of claim 11, wherein R^(b) and R^(d) areindependently hydrogen or methyl.
 13. The compound of claim 12, whereinR^(e) is C₁₋₄ alkylene.
 14. The compound of claim 13, wherein R^(c) is—Y′—R″—Y′—, where each Y′ is a direct bond and R″ is C₁₋₁₂ alkylene. 15.The compound of claim 1, wherein R¹ and R² are hydrogen.
 16. A compoundof formula II:

or a pharmaceutically acceptable salt thereof; wherein W is N or CCl; R⁹is selected from the group consisting of hydrogen, C₁₋₆ alkyl and C₃₋₆cycloalkyl, where alkyl and cycloalkyl are optionally substituted with—COOH or 1 to 3 fluoro substituents; the pyridinium ring has meta orpara substitution; R¹⁰ is hydrogen, C₁₋₄ alkyl or C₂₋₄ alkenyl; R¹¹ isC₁₋₁₂ alkylene or C₂₋₁₂ alkenylene; and R¹² is hydrogen, C₁₋₄ alkyl orC₂₋₄ alkenyl.
 17. The compound of claim 16, wherein W is CCl.
 18. Thecompound of claim 16, wherein R¹⁰ and R¹² are hydrogen or methyl. 19.The compound of claim 16, wherein R¹¹ is C₁₋₁₀ alkylene.
 20. Thecompound of claim 16, wherein W is CCl; R⁹ is methyl; R¹⁰ is hydrogen;R¹¹ is —(CH₂)₄—; R¹² is hydrogen; and the pyridinium ring is parasubstituted.
 21. The compound of claim 16, wherein the compound isselected from: Pyridinium R⁹ R¹⁰ R¹¹ R¹² W Substitution —CH₃ —H—(CH₂)₁₀— —H CCl para —CH₃ —H —(CH₂)₄— —H CCl para —CH₃ —H —(CH₂)₄— —HCCl meta —CH₃ —H —CH₂— —H CCl para —CH₃ —H —CH₂— —H CCl meta —CH₃ —H—(CH₂)₂— —H CCl para —CH₃ —H —(CH₂)₂— —H CCl meta —CH₃ —H —(CH₂)₃— —HCCl para —CH₃ —H —(CH₂)₃— —H CCl meta —CH₃ —H —(CH₂)₅— —H CCl para —CH₃—H —(CH₂)₅— —H CCl meta —CH₃ —H —(CH₂)₆— —H CCl para —CH₃ —H —(CH₂)₆— —HCCl meta —CH₃ —H —(CH₂)₇— —H CCl para —CH₃ —H —(CH₂)₇— —H CCl meta —CH₃—H —(CH₂)₈— —H CCl para —CH₃ —H —(CH₂)₈— —H CCl meta —CH₃ —H —(CH₂)₉— —HCCl para —CH₃ —H —(CH₂)₉— —H CCl meta —CH₃ —H —(CH₂)₁₀— —H CCl meta —CH₃—H —(CH₂)₁₁— —H CCl para —CH₃ —H —(CH₂)₁₁— —H CCl meta —CH₃ —H —(CH₂)₁₂——H CCl para —CH₃ —H —(CH₂)₁₂— —H CCl meta —CH₂CH₃ —H —(CH₂)₄— —H CClpara —CH₂CH₃ —H —(CH₂)₄— —H CCl meta —OH —H —(CH₂)₄— —H CCl para —OH —H—(CH₂)₄— —H CCl meta —C(CH₃)₂COOH —H —(CH₂)₄— —H CCl para —C(CH₃)₂COOH—H —(CH₂)₄— —H CCl meta —CH₂CH₂F —H —(CH₂)₄— —H CCl para —CH₂CH₂F —H—(CH₂)₄— —H CCl meta -cyclopentyl —H —(CH₂)₄— —H CCl meta -cyclopentyl—H —(CH₂)₄— —H CCl para —CH₃ —CH₃ —(CH₂)₄— —H CCl para —CH₃ —CH₃—(CH₂)₄— —H CCl meta —CH₃ —H —(CH₂)₄— —CH₃ CCl para —CH₃ —H —(CH₂)₄——CH₃ CCl meta —CH₃ —H —(CH₂)₄— —H N para —CH₃ —H —(CH₂)₄— —H N meta—CH₂CH₃ —H —(CH₂)₄— —H N para —CH₂CH₃ —H —(CH₂)₄— —H N meta —OH —H—(CH₂)₄— —H N para —OH —H —(CH₂)₄— —H N meta —C(CH₃)₂COOH —H —(CH₂)₄— —HN para —C(CH₃)₂COOH —H —(CH₂)₄— —H N meta —CH₂CH₂F —H —(CH₂)₄— —H N para—CH₂CH₂F —H —(CH₂)₄— —H N meta -cyclopentyl —H —(CH₂)₄— —H N meta-cyclopentyl —H —(CH₂)₄— —H N para —CH₃ —CH₃ —(CH₂)₄— —H N para —CH₃—CH₃ —(CH₂)₄— —H N meta —CH₃ —H —(CH₂)₄— —CH₃ N para —CH₃ —H —(CH₂)₄——CH₃ N meta


22. A pharmaceutical composition comprising a pharmaceuticallyacceptable carrier and a therapeutically effective amount of a compoundof any one of claims 1 to
 21. 23. A method of treating a bacterialinfection in a mammal, the method comprising administering to a mammal apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a therapeutically effective amount of a compound of any oneof claims 1, 16 or
 21. 24. A method of inhibiting the growth ofbacteria, the method comprising contacting bacteria with agrowth-inhibiting amount of a compound of any one of claims 1, 16 or 21.25. A method of inhibiting bacterial cell wall biosynthesis, the methodcomprising contacting bacteria with a cell wall biosynthesis-inhibitingamount of a compound of any one of claims 1, 16 or
 21. 26. A process forpreparing compound of formula I:

or a salt or protected derivative thereof; wherein each of X¹ and X² isindependently hydrogen or chloro; W is N or CCl; R¹ and R² areindependently selected from hydrogen and C₁₋₆ alkyl; each R³ isindependently selected from C₁₋₆ alkyl, OR, halo, —SR, —S(O)R, —S(O)₂R,and —S(O)₂OR, where each R is independently C₁₋₆ alkyl optionallysubstituted with COOH or 1 to 3 fluoro substituents; one of R⁴ and R⁵ ishydroxy and the other is hydrogen; R⁶ and R⁷ are independently hydrogenor methyl; R⁸ is hydrogen or a group of the formula:

R⁹ is selected from hydrogen, C₁₋₆ alkyl and C₃₋₆ cycloalkyl, wherealkyl and cycloalkyl are optionally substituted with —COOH or 1 to 3fluoro substituents; R^(a) is —Y—R″—, where R″ is selected from C₁₋₁₂alkylene, C₂₋₁₂ alkenylene, C₂₋₁₂ alkynylene, C₃₋₆ cycloalkylene, C₆₋₁₀arylene, C₂₋₉ heteroarylene, C₃₋₆ heterocycle and combinations thereof,and is optionally substituted with 1 or 2 groups selected from Z, whereeach Z is independently selected from the group consisting of —OR′,—SR′, —F, —Cl, —N(R′)₂, —OC(O)R′, —C(O)OR′, —NHC(O)R′, —C(O)N(R′)₂,—CF₃, —OCF₃, and side chains of naturally-occurring amino acids, whereeach R′ is independently hydrogen or C₁₋₄ alkyl; and R″ contains at most20 non-hydrogen atoms; and Y, which links R″ to the pyridinium ring at ameta or para position, is selected from a direct bond, NR′, O, S, C(O),NR′C(O), and C(O)NR′, precluding direct bonds between heteroatoms in Yand R″; each R^(b) and R^(d) is independently selected from hydrogen,C₁₋₆ alkyl, C₂₋₆ alkenyl and C₂₋₆ alkynyl; each R^(c) is independently adirect bond or —Y′—R″—Y′—, where each Y′ is independently selected froma direct bond, O and NR′, precluding direct bonds between heteroatoms inY′ and R″; each R^(e) is independently selected from the group definedby R″; n is an integer from 0 to 3; x is an integer from 0 to 2; and yis an integer from 0 to 2: or a salt thereof, the process comprisingcomprising reacting a compound of formula 1:

or a salt, or an activated and/or protected derivative thereof, with acompound of formula 2:

or a salt or carboxy-protected derivative thereof; in the presence of acompound of formula 3:HOOC—R^(c)—COOH  3 or a salt, activated derivative, or protectedderivative thereof; to form the compound of formula I, or a salt orprotected derivative thereof.
 27. The process of claim 26, wherein theprocess further comprises forming a pharmaceutically acceptable salt ofthe compound of formula I.
 28. The product prepared by the process ofclaim 26 or 27.